European Urology Oncology

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European Urology Oncology Combined Next-generation Sequencing and Flow Cytometry Analysis for an Anti-PD-L1 Partial Responder over Time: An Exploration of Mechanisms of PD-L1 Activity and Resistance in Bladder Cancer Max Kates, Thomas R. Nirschl, Alex S. Baras, Nikolai A. Sopko, Noah M. Hahn, Xiaoping Su, Jiexin Zhang, Christina M. Kochel, Woonyoung Choi, David J. McConkey, Charles G. Drake, Trinity J. Bivalacqua European Urology Oncology DOI: 10.1016/j.euo.2019.01.017 Copyright © 2019 European Association of Urology Terms and Conditions

Fig.1 Fresh tissue was obtained from early and late PD-L1 resistance time points for flow cytometry to specifically assess protein expression of immune checkpoints. A) T Cell sub-populations, including effector CD4+ (Tconv), regulatory CD4+ (Treg), CD8+ and B) total CD3+ cells were assessed. C) Protein expression of PD-1, LAG-3, TIM-3, and CTLA-4 were assessed on CD8+, CD4+FOXP3-, (Teff) and CD4+FOXP3+(Tregs) cells at both timepoints. D) Total number of checkpoint co-expression on CD8, CD4 effector, and CD4 Tregs were analyzed. European Urology Oncology DOI: (10.1016/j.euo.2019.01.017) Copyright © 2019 European Association of Urology Terms and Conditions

Fig. 2 Gene expression of select immune markers across all timepoints demonstrates increased expression of FOXP3, HAVCR2 (TIM-3), and CTLA-4 as the patient became PD-L1 resistant. European Urology Oncology DOI: (10.1016/j.euo.2019.01.017) Copyright © 2019 European Association of Urology Terms and Conditions